The objective of my PhD study was to develop and characterize new methods and
sensors based on fluorescence resonance energy transfer (FRET) for bioanalysis.
Chapter 3 describes the use of FRET between donor fluorophores and acceptor labeled
murine macrophage cells. FRET microscopy was used to determine whether the donor
molecules truly permeate through the cell membrane or only adsorb to the cell surface.
This method was found to be partially successful since the donor red tail fluorescence
overlapped with the sensitized acceptor fluorescence and led to false reading of FRET.
We found that is easier to monitor delivery of acceptor molecules into donor-labeled
cells. Using donor labeled cells it was possible to determine whether the acceptor
molecules were actually delivered into cells. However, a relatively high acceptor
concentration in the hundreds of micromolar level was needed to obtain measurable
FRET signals in the 3-D cellular system. The results underscored the need to reduce
the dimensionality of FRET systems in order to increase the FRET efficiency between
donor and acceptor molecules.
Chapter 4 describes the development of FRET sensing lipobeads labeled with donors
and their use to evaluate the interactions of acceptor molecules with the phospholipid
membrane of FRET sensing lipobeads. The change in the dimensionality of the system
in which FRET occurs, improved the sensitivity of our measurements by 3-folds
compared to FRET measurements in solution. We concluded that a molecular
recognition component had to be added to the sensing particles to further increase their
selectivity and sensitivity. Chapter 5 describes the development of FRET trap sensing beads and their use for
screening nonfluorescent carbohydrates and glycoproteins. The FRET sensing
technique was based on binding between dextran molecules labeled with Texas Red
(Dextran-TR) and polystyrene microparticles labeled with Fluorescein tagged
Concanavalin A (FITC-ConA). It was found that carbohydrates and glycoproteins inhibit
the binding between dextran-TR and FITC-ConA labeled particles. The inhibition effect
was concentration dependent thus enabled screening carbohydrates and glycoproteins
based on their inhibition potency. The dissertation critically evaluates the performance
of FRET microscopy and FRET based sensors in delivery and screening applications.

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